Herpesviruses are ubiquitous and are responsible for many important human diseases. A better molecular understanding of how they replicate is essential for finding ways to fight them. Perhaps the least understood herpesviral proteins are those of the tegument, the region of the virion located between the capsid and the envelope. These proteins help drive the virus assembly and budding events in the infected cell, but they also play numerous other roles in virus replication. The focus of this competing continuation application is UL16, a poorly understood tegument protein of herpes simplex virus that is conserved among all the herpes viruses (alpha, beta, and gamma). The goal of the previous funding period was to elucidate the direct- interaction network of UL16 (i.e., which proteins does UL16 actually touch). The Wills and Semmes labs made a lot of progress, but there were two remarkable observations that provide the foundation for this proposal. First, while examining its capsid-association properties, we discovered that UL16 is released when the virus engages its attachment receptors on the surface of the host cell. This occurs before fusion and even when the receptors are immobilized on agarose beads. This is the first example of an external signal being transmitted to the interior of an enveloped virus. Second, we discovered that UL16 interacts specifically and directly with the cytoplasmic tail of gE, a viral protein that is essental for cell-to-cell spread. To pursue these major clues about the role(s) of UL16 in the HSV replication cycle, we have devised a comprehensive set of four aims that will further define it functions. The first goal is to dissect the molecular organization and regulatory mechanism of UL16. We hypothesize that eight conserved cysteines in the C-terminal regulatory domain are key to controlling the N-terminal binding domain that interacts with other viral proteins. The second goal is to elucidate the mechanism and purpose of the signal-mediated tegument rearrangement. We hypothesize that the signal is used either to activate the fusion machinery or to initiate virus UN coating. The third goal is to determine the role of proteins in the UL16 interaction network in the mechanism of cell-to-cell spread. We hypothesize that their binding with the tail of gE promotes lateral spread, perhaps in conjunction with host protein Rich1, which we found to be present in native complexes with UL16. The fourth goal is to elucidate the localization mechanism and function of UL16 in the nucleus. It has long been known that this tegument protein accumulates there, but nothing is known about how it gets in or what it binds to there. These four aims are important because UL16 is clearly involved in multiple events in the virus replication cycle, which means that drugs that interfere with those function may cause multiple blocks.